An edible mushroom substrate standard quality control sample containing multiple arsenic forms and a preparation method and application thereof

Abstract

The application provides an edible mushroom substrate standard quality control sample containing multiple arsenic forms and a preparation method and application thereof, and relates to the technical field of standard substances. The edible mushroom dry sample (containing monomethylarsonic acid, dimethylarsinic acid, arsenobetaine and arsenite) is made into powder to obtain a powder sample; an arsenate aqueous solution is added to the powder sample, and the obtained damp powder sample is sequentially dried, crushed and mixed to obtain the edible mushroom substrate standard quality control sample containing multiple arsenic forms. The edible mushroom dry sample is selected as a raw material, the prepared edible mushroom substrate standard quality control sample is easy to store stably, and the temporary sampling process is not easy to cause the transformation of arsenic forms. The method is simple, the prepared edible mushroom substrate standard quality control sample has good uniformity and excellent stability, can be used for accurate quantification of inorganic arsenic of edible mushrooms, and can effectively prevent the false positive situation of inorganic arsenic results through qualitative determination of organic arsenic, and effectively improves the accuracy and reliability of the detection results.

Description

Technical Field

[0001] This invention relates to the field of standard material technology, and in particular to a standard quality control sample of edible fungi matrix containing multiple arsenic forms, its preparation method, and its application. Background Technology

[0002] The distribution of arsenic speciation varies significantly among different food categories. For example, arsenic in seafood is mainly in the form of low-toxicity organic arsenic (such as arsenic betaine), while arsenic in grains is primarily in the form of highly toxic inorganic arsenic. The distribution of arsenic speciation in edible fungi is more complex, with high levels of arsenic betaine, monomethylarsenic acid, and dimethylarsenic acid in addition to inorganic arsenic. During detection, improper chromatographic conditions can easily lead to overlapping peaks, potentially resulting in false positive results.

[0003] Currently, there are no commercially available inorganic arsenic quality control samples for food matrices, nor are they being used on a large scale. The main reasons are twofold: First, existing inorganic arsenic quality control samples for food matrices only involve fresh sample preparation, which makes sample preservation and operation difficult for users, and inconvenient for practical application. Second, current quality control samples do not include organic arsenic forms, which means that in actual testing, both organic and inorganic arsenic will produce significant responses under the same chromatographic conditions. Due to the lack of effective quality control methods, it is difficult to eliminate false positive interference, thus significantly reducing the actual effectiveness of quality control. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a standard quality control sample for edible fungi substrate containing multiple arsenic forms, its preparation method, and its application. The standard quality control sample for edible fungi substrate containing multiple arsenic forms provided by this invention is easy to preserve stably, can be used for accurate quantification of inorganic arsenic in edible fungi, and effectively prevents false positives in inorganic arsenic results through qualitative analysis of organic arsenic.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing standard quality control samples of edible fungi substrate containing multiple arsenic forms, comprising the following steps:

[0007] The dried edible fungi sample was made into powder to obtain a powder sample; the dried edible fungi sample was shiitake mushroom, porcini mushroom or matsutake mushroom, and the dried edible fungi sample contained monomethylarsine, dimethylarsine, arsenoside betaine and arsenite, and the content of arsenoside betaine in the dried edible fungi sample was not less than 0.01 mg / kg, the content of monomethylarsine was 0.01~0.1 mg / kg, the content of dimethylarsine was 0.01~0.1 mg / kg, and the content of arsenite was not less than 0.02 mg / kg;

[0008] An aqueous solution of arsenate was added to the powder sample to obtain a moist powder sample;

[0009] The wet powder sample was dried, pulverized and mixed in sequence to obtain the standard quality control sample of edible fungus substrate containing multiple arsenic forms. The content of inorganic arsenic in the standard quality control sample of edible fungus substrate containing multiple arsenic forms was 0.6~1.2 mg / kg. The contents of monomethylarsenic acid, dimethylarsenic acid, arsenic betaine, arsenite and inorganic arsenic were all calculated as elemental arsenic.

[0010] Preferably, the method for preparing the powder includes:

[0011] After removing the base of the stem from the dried edible fungi sample, it was cut into pieces, crushed, and sieved in sequence to obtain a powder sample; the sieve used for sieving had a mesh size of 0.15 mm.

[0012] Preferably, the concentration of the arsenate aqueous solution is 0.5~1 mg / L, and the concentration is calculated as arsenic element; the mass ratio of the powder sample to the volume of the arsenate aqueous solution is 1 g:(0.8~1.2) mL.

[0013] Preferably, the method of adding an aqueous arsenate solution to the powder sample includes:

[0014] The powder sample is laid up in a layer, and the arsenate aqueous solution is sprayed onto the layer. After stirring and standing, the moist powder sample is obtained.

[0015] Preferably, the thickness of the layup is 0.3~0.6mm; the spraying pressure is 0.2~0.4MPa and the height is 20~40cm; the stirring rate is 30~100r / min and the time is 15~30min; and the settling time is 120~240min.

[0016] Preferably, the drying process includes performing a first drying and a second drying in sequence. The temperature of the first drying is 50-60°C and the time is 4-6 hours. The temperature of the second drying is 100-105°C and the time is 0.5-1 hours.

[0017] Preferably, after crushing, the pulverized material is further sieved, and the sieve used has a mesh size of 0.15 mm.

[0018] Preferably, the mixing is carried out in a high-efficiency mixer, the high-efficiency mixer having a working speed of 40~50 r / min and a mixing time of 6~8 h.

[0019] This invention provides standard quality control samples of edible fungi substrate containing multiple arsenic forms prepared by the preparation method described above.

[0020] This invention provides the application of the standard quality control sample of edible fungi matrix containing multiple arsenic forms described in the above technical solution in the detection of inorganic arsenic content in edible fungi products.

[0021] This invention provides a method for preparing a standard quality control sample of edible fungi matrix containing multiple arsenic forms. The method uses dried edible fungi samples containing arsenite [As(III)] at a background level not lower than the limit of quantitation (0.02 mg / kg) and three organic arsenic compounds (monomethylarsenic acid, dimethylarsenic acid, and arsenic betaine) as raw materials. The content of monomethylarsenic acid and dimethylarsenic acid is not higher than 0.1 mg / kg to effectively control the long-term stability of organic arsenic on inorganic arsenic determination. The dried edible fungi sample is then powdered, and pentavalent arsenic [As(V)] is added. After drying, pulverizing, and mixing, a standard quality control sample of edible fungi matrix containing five arsenic forms (three organic arsenic compounds and two inorganic arsenic compounds [As(V)] and [As(III)]) is obtained. Compared with the prior art, this invention has the following beneficial effects:

[0022] This invention uses dried edible fungi samples as raw materials. The prepared edible fungi matrix standard quality control samples are easy to store and readily available. Furthermore, the temporary sampling process is less likely to cause the transformation of arsenic forms.

[0023] The preparation method of this invention is simple, and the prepared edible fungi substrate standard quality control samples have good uniformity and excellent stability (under low temperature storage conditions of 4℃, the opened samples can maintain stability for up to 12 months).

[0024] The edible fungi matrix standard quality control sample prepared by this invention can be used for the accurate quantification of inorganic arsenic in edible fungi, and effectively prevents false positives in inorganic arsenic results through the qualitative analysis of organic arsenic. When determining the inorganic arsenic content according to GB 5009.11-2024 "National Food Safety Standard - Determination of Total Arsenic and Inorganic Arsenic in Food", the chromatographic separation step must achieve complete separation of the chromatographic peaks of organic and inorganic arsenic. If the chromatographic peaks overlap, the measured result will be too high, leading to a risk of false positives. The standard quality control sample prepared by this invention covers common organic arsenic forms in food (monomethylarsenic acid, dimethylarsenic acid, and arsenic betaine). Its matrix is ​​derived from screened natural background samples, rather than being added later, thus exhibiting good stability. When used for quality control, this standard quality control sample can be measured simultaneously with the sample to be tested. If the organic arsenic chromatographic peak of the quality control sample can be completely separated from the inorganic arsenic, the separation accuracy of the sample to be tested under the same conditions can be ensured, thereby achieving qualitative quality control and effectively avoiding false positive results.

[0025] Furthermore, the edible fungi substrate standard quality control samples prepared by this invention can achieve precise quantitative quality control of samples exceeding the limit: GB 2762-2025 "National Food Safety Standard - Limits of Contaminants in Food" specifies limits for inorganic arsenic in different foods ranging from 0.1 to 0.8 mg / kg. In the design of the standard quality control samples, this invention specifically targets the target limit (especially in cases of exceeding the limit) for result quality control. By adjusting the preparation conditions and the concentration of additives, the concentration of the prepared standard quality control samples is precisely set near the target limit. When a sample exceeds the limit, the standard quality control material obtained by this method can be measured simultaneously with the sample, thereby achieving targeted quality control of the results for samples exceeding the limit, effectively avoiding misjudgments of results exceeding the limit, and reducing laboratory risks.

[0026] In summary, the standard quality control samples prepared by this invention have important application value in the detection of inorganic arsenic content in food, and can effectively improve the accuracy and reliability of the detection results. Attached Figure Description

[0027] Figure 1 The dynamic changes in the content of trivalent arsenic As(III) (a) and pentavalent arsenic As(V) (b) were observed.

[0028] Figure 2 Chromatogram of the standard solution;

[0029] Figure 3 The chromatogram of the inorganic arsenic standard control sample in the prepared edible fungus matrix is ​​shown. Detailed Implementation

[0030] This invention provides a method for preparing standard quality control samples of edible fungi substrate containing multiple arsenic forms, comprising the following steps:

[0031] The dried edible fungi were ground into powder to obtain a powder sample. The dried edible fungi were shiitake mushrooms, porcini mushrooms, or matsutake mushrooms. The dried edible fungi contained monomethylarsine, dimethylarsine, arsenoside betaine, and arsenite. The content of arsenoside betaine in the dried edible fungi was not less than 0.01 mg / kg (limit of quantitation), the content of monomethylarsine was 0.01 (limit of quantitation) to 0.1 mg / kg, the content of dimethylarsine was 0.01 (limit of quantitation) to 0.1 mg / kg, and the content of arsenite was not less than 0.02 mg / kg.

[0032] An aqueous solution of arsenate was added to the powder sample to obtain a moist powder sample;

[0033] The wet powder sample was dried, pulverized and mixed in sequence to obtain the standard quality control sample of edible fungus substrate containing multiple arsenic forms. The content of inorganic arsenic in the standard quality control sample of edible fungus substrate containing multiple arsenic forms was 0.6~1.2 mg / kg. The contents of monomethylarsenic acid, dimethylarsenic acid, arsenic betaine, arsenite and inorganic arsenic were all calculated as elemental arsenic.

[0034] Unless otherwise specified, all raw materials involved in this invention are commercially available products well known in the art.

[0035] This invention prepares dried edible fungi samples into powder to obtain powder samples.

[0036] This invention uses dried edible fungi samples as raw materials. Its advantages lie in the fact that, after preparation, they can be stably stored at 4°C, the storage conditions are simple, they are easy to access at any time, and the temporary sampling process is less likely to induce arsenic transformation. Fresh edible fungi, such as fresh shiitake mushrooms, have a high water content (up to 80%), and to prevent spoilage, they need to be frozen at -18°C. However, this poses significant difficulties for daily sample access, requiring long thawing times before use. Repeated freeze-thaw cycles can easily lead to arsenic transformation. Furthermore, thawed samples, due to their high water content, are prone to stratification, resulting in decreased sample homogeneity.

[0037] In this invention, the dried edible fungus sample is shiitake mushroom, porcini mushroom, or matsutake mushroom. In this invention, the dried edible fungus sample contains monomethylarsenic acid (MMA), dimethylarsenic acid (DMA), arsenobetaine (AsB), and arsenite [As(III)], wherein the content of arsenobetaine in the dried edible fungus sample is not less than 0.01 mg / kg, the content of monomethylarsenic acid and dimethylarsenic acid is between the limit of quantitation (0.01 mg / kg) and 0.1 mg / kg, and the content of arsenite is not less than 0.02 mg / kg (the 0.02 mg / kg is the limit of quantitation for arsenite [As(III)]). In embodiments of this invention, the shiitake mushroom can be from Zhejiang, Yunnan, or Jilin; the porcini mushroom can be from Yunnan; and the matsutake mushroom can be from Zhejiang.

[0038] This invention uses dried edible fungi samples containing arsenic betaine, monomethylarsenic acid, and dimethylarsenic acid in the background as raw materials to facilitate accurate determination of chromatographic peaks and avoid false positives in the quality control process for inorganic arsenic (As(III) and As(V)). Considering the stability of monomethylarsenic acid and dimethylarsenic acid in plant systems, which may transform into inorganic arsenic under certain conditions, the content of monomethylarsenic acid and dimethylarsenic acid in the sample is limited to no more than 0.1 mg / kg. Furthermore, since arsenic choline (AsC) is usually present in extremely low amounts or undetectable in the sample, and its chromatographic peak position is unlikely to cause false positive interference in the determination of inorganic arsenic, the content of this arsenic form does not need to be specified. Arsenic betaine is chemically stable in fungal samples and does not easily undergo form transformation under room temperature conditions; therefore, this invention only requires that the content of arsenic betaine not be lower than the limit of quantitation. If organic arsenic (monomethylarsenic acid and dimethylarsenic acid) is introduced by addition, both will undergo significant degradation and transformation in a short period of time, resulting in unstable physicochemical properties of the standard quality control sample.

[0039] This invention also requires that the raw material of dried edible fungi contain arsenite [As(III)] at a level not lower than the limit of quantitation (0.02 mg / kg). This is because if As(III) is introduced by addition, it is easy for As(III) to convert to As(V), resulting in a change in the distribution of arsenic speciation. This speciation instability may affect the long-term validity of the standard quality control sample and is not conducive to the user's subsequent stable use. However, when As(V) is introduced by addition, the content of As(V) does not change significantly with the extension of storage time; therefore, this invention introduces As(V) into the sample in the form of an additive.

[0040] In this invention, the method for preparing the powder preferably includes:

[0041] After removing the base of the stem from the dried edible fungi sample, it was cut into pieces, crushed, and sieved in sequence to obtain a powder sample.

[0042] In this invention, the shiitake mushrooms, after the base of the stem has been removed, can be processed into small pieces less than 1 cm by manual shearing or mechanical cutting. In this invention, the crushing can be carried out using a pulverizer. In this invention, the sieve used for sieving preferably has a mesh size of 0.15 mm, and after sieving, a uniform powder sample is obtained.

[0043] After obtaining the powder sample, the present invention adds an aqueous solution of arsenate to the powder sample to obtain a moist powder sample.

[0044] In this invention, the amount of arsenate aqueous solution added is determined to ensure that the content of inorganic arsenic (i.e., As(III) and As(V)) in the final edible fungus substrate standard quality control sample containing multiple arsenic forms is 0.6~1.2 mg / kg. In embodiments of this invention, the concentration of the arsenate aqueous solution is preferably 0.5~1 mg / L, and can be 0.5, 0.6, 0.8, or 1 mg / L; the arsenate aqueous solution is preferably prepared using ultrapure water, and serves as an additive solution for inorganic arsenic (pentavalent arsenic [As(V)]); the mass ratio of the powder sample to the volume of the arsenate aqueous solution is preferably 1 g:(0.8~1.2) mL. By controlling the mass ratio of the powder sample to the volume of the arsenate aqueous solution within the above range, this invention effectively forms a loose, non-clumping, moist powder.

[0045] In this invention, the method of adding an aqueous arsenate solution to a powder sample preferably includes:

[0046] The powder sample is laid up in a layer, and the arsenate aqueous solution is sprayed onto the layer. After stirring and standing, the moist powder sample is obtained.

[0047] In this invention, the thickness of the layup is preferably 0.3~0.6 mm, and can be 0.3, 0.4, 0.5 or 0.6 mm. The powder sample can be sieved into a glass container in batches using a sieve with a pore size of 0.15 mm to form a layup with a thickness of 0.3~0.6 mm for each batch of sample. In this invention, the spraying can be done using an electric sprayer, the spraying pressure is preferably 0.2~0.4 MPa, and the spraying height is preferably 20~40 cm, and can be 20, 30 or 40 cm. In an embodiment of this invention, the spraying process specifically involves dividing the work area into several independent areas, maintaining sufficient isolation between the areas to avoid cross-interference; after each area has completed its work independently, the samples are then combined; the continuous spraying time in each area can be 5~10 seconds.

[0048] In this invention, the stirring rate is preferably 30~100 r / min, and the stirring time is preferably 15~30 min. Specifically, the stirring can be carried out by placing the sample after spraying the arsenate aqueous solution in a vertical mixer. The settling time is preferably 120~240 min, which can be 120, 150 or 200 min. After settling, a uniform moist powder sample is formed.

[0049] After obtaining the wet powder sample, the present invention sequentially dries, pulverizes and mixes the wet powder sample to obtain the standard quality control sample of edible fungus substrate containing multiple arsenic forms.

[0050] In this invention, the drying process preferably includes a first drying and a second drying in sequence. The temperature of the first drying is preferably 50-60°C, and the time is preferably 4-6 hours. The temperature of the second drying is preferably 100-105°C, and the time is preferably 0.5-1 hours. In an embodiment of this invention, the wet powder sample is placed in a stainless steel tray, spread into a thin layer of 2-3 cm thickness, and then placed in a forced-air drying oven for drying. This invention gently evaporates the free water on the sample surface through the first drying, and thoroughly kills microorganisms (such as mold and bacteria) and removes residual bound water through the second drying, ensuring that the sample will not deteriorate due to microbial activity during subsequent storage.

[0051] In this invention, the pulverization is specifically carried out in a pulverizer; after pulverization, it is preferable to further sieve the resulting pulverized material and collect the undersize material; the sieve mesh size is preferably 0.15 mm. In this invention, the mixing is preferably carried out in a high-efficiency mixer, the operating speed of the high-efficiency mixer is preferably 40~50 r / min, and the mixing time is preferably 6~8 h.

[0052] After mixing, the present invention preferably encapsulates the obtained edible fungus substrate standard quality control sample in a glass bottle with nitrogen filling and stores it in a refrigerator at 4°C; the nitrogen purity used for nitrogen filling and encapsulation is preferably ≥99.99%, the nitrogen filling pressure is preferably 0.05~0.1MPa, and the replacement number is preferably 2~3 times.

[0053] The preparation method provided by this invention is simple to operate, and the prepared samples have good uniformity and excellent stability. It has important application value in the detection of inorganic arsenic content in food and can effectively improve the accuracy and reliability of the detection results.

[0054] This invention provides standard quality control samples of edible fungi substrate containing multiple arsenic forms prepared by the preparation method described above.

[0055] This invention provides the application of the standard quality control sample of edible fungi matrix containing multiple arsenic forms described in the above technical solution in the detection of inorganic arsenic content in edible fungi products.

[0056] In this invention, the edible fungi product can be shiitake mushrooms, porcini mushrooms, matsutake mushrooms, etc. This invention does not specifically limit the method for detecting the inorganic arsenic content in edible fungi products using the standard quality control sample containing multiple arsenic forms of edible fungi matrix; conventional detection methods in the art are sufficient. The detection conditions corresponding to the method should be such that the organic and inorganic arsenic peaks in the edible fungi matrix standard quality control sample are completely separated. In this embodiment of the invention, the detection method is liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS / MS). The HPLC conditions include: Hamilton PRPX100 anion exchange column (4.1×250mm, 5μm), column temperature 25℃; injection volume 10μL; gradient elution program; mobile phase A is an aqueous solution containing 2% (volume percentage) methanol; mobile phase B is a 50mM ammonium carbonate ((NH4)2CO3) solution; and the elution program is shown in Table 2. The ICP-MS / MS conditions include: RF power 1500W, plasma gas (Ar) flow rate 15L / min, reaction gas (O2) flow rate 0.8mL / min, and nebulizer gas flow rate 1.0L / min.

[0057] The standard quality control samples of edible fungi matrix containing multiple arsenic forms provided by this invention can fully meet the quality control requirements for the detection of inorganic arsenic in edible fungi products, ensure that false positive results are effectively avoided, and achieve the accuracy of quantitative analysis.

[0058] To further illustrate the present invention, the following detailed description, in conjunction with examples, of the standard quality control samples of edible fungi substrates containing multiple arsenic forms provided by the present invention, their preparation methods, and applications, should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] (a) Selection of raw materials

[0061] Previous experimental results showed that when organic arsenic (monomethylarsenic acid (MMA) and dimethylarsenic acid (DMA)) was introduced using the additive method, both underwent significant degradation and transformation within a short period of time, resulting in unstable physicochemical properties of the standard quality control samples. Therefore, it was impossible to obtain stable and compliant organic arsenic-containing samples using the additive method.

[0062] This invention determined the total arsenic content and arsenic speciation in different types of edible fungi. During sample screening, in addition to inorganic arsenic (As(III) and As(V)), the presence of arsenic betaine (AsB), monomethylarsenic acid (MMA), and dimethylarsenic acid (DMA) in the background was used as a standard to facilitate accurate peak characterization and avoid false positives for inorganic arsenic (As(III) and As(V)) during quality control. Considering the stability of MMA and DMA in plant systems, which may transform into inorganic arsenic under certain conditions, the content of MMA and DMA in the samples was limited to no more than 0.1 mg / kg. Furthermore, since arsenic choline (AsC) is usually present in very low amounts or undetectable in samples, and its chromatographic peak position is unlikely to cause false positive interference in the determination of inorganic arsenic, the content of this arsenic form does not need to be specified; while arsenic betaine (AsB) is chemically stable in fungal samples and does not easily undergo form transformation under room temperature conditions, therefore, the content of this arsenic form only needs to be not lower than the limit of quantitation (0.01 mg / kg).

[0063] Table 1 lists the screening results of arsenic forms in common edible fungi. The contents of different arsenic forms in Table 1 are all calculated as elemental arsenic.

[0064] Table 1. Test results (mg / kg) of edible fungi samples of different types / origins.

[0065]

[0066] Table 1 shows that AsB was not detected in oyster mushrooms, wood ear mushrooms, and king oyster mushrooms, and DMA and MMA were not detected in many samples, making them unsuitable as raw materials for standard quality control samples. Shiitake mushrooms and tea tree mushrooms have a relatively complete range of arsenic speciation. Among them, shiitake mushrooms, porcini mushrooms, and matsutake mushrooms were found to contain all five arsenic speciations except AsC (AsB, MMA, DMA, As(III), and As(V)), and the concentrations of each speciation met the requirements of the experimental design. Based on the above analysis, shiitake mushrooms, porcini mushrooms, and matsutake mushrooms meet the requirements; however, considering the sample preparation cost, this invention ultimately selected Jilin shiitake mushrooms as the raw material for preparing arsenic speciation standard quality control samples.

[0067] (ii) Selection of additives

[0068] This invention presents an optimal addition experiment for two forms of inorganic arsenic (As(III) and As(V)). Standard solutions of As(III) and As(V) were added to shiitake mushroom powder samples, respectively, and processed according to standard quality control sample preparation procedures. The prepared samples were stored at 4°C under normal sealed conditions (without nitrogen purging, simulating actual usage conditions after opening) to investigate the changing trends of arsenic speciation over time under the two addition methods.

[0069] Figure 1This describes the dynamic changes in the content of trivalent arsenic As(III) (a) and pentavalent arsenic As(V) (b). Through... Figure 1 The time-series monitoring results show that the standard control sample with added As(III) exhibits a rapid oxidation conversion rate after preparation (day 0), with most of it being oxidized to As(V), and this conversion process continues with prolonged storage time. From the perspective of the stability of the standard control sample, although the total amount of inorganic arsenic in the sample did not change significantly, As(III) readily converts to As(V), leading to a change in the distribution of arsenic speciation. This speciation instability may affect the long-term effectiveness of the standard control sample, which is detrimental to its subsequent stable use by users. In the standard control sample with added As(V), the contents of As(III) and As(V) did not change significantly with prolonged storage time after preparation. Therefore, this invention prefers As(V) as the inorganic arsenic additive. Furthermore, according to GB 2762-2025 "National Food Safety Standard - Limits of Contaminants in Food," the maximum limit for edible fungi and their products is 0.8 mg / kg. The final standard substance designed in this invention has an inorganic arsenic concentration range of 0.6~1.2 mg / kg to more effectively support the accurate determination of samples exceeding the limit.

[0070] (III) Preparation process

[0071] The preparation of a standard quality control sample for edible fungi substrate containing multiple arsenic forms (also known as an inorganic arsenic standard quality control sample for edible fungi substrate) uses shiitake mushrooms as raw material. The process involves crushing, adding inorganic arsenic, homogenization, stepwise drying, mixing, nitrogen filling, and packaging. The specific steps are as follows:

[0072] S1. Using dried shiitake mushrooms as raw material, arsenic speciation was first determined by HPLC-ICP-MS / MS. The contents of MMA and DMA were not higher than 0.1 mg / kg.

[0073] S2. After removing the base of the stipe, the sample is processed into small pieces less than 1 cm by manual shearing or mechanical cutting. Then, it is further crushed by a pulverizer. The processed sample is sieved through a sieve with a sieve hole diameter of 0.15 mm to obtain a uniform powder sample.

[0074] S3. Use ultrapure water to prepare an arsenate solution as an inorganic arsenic additive solution with a concentration of 0.8 mg / L for addition to the powder.

[0075] S4. The powder was sieved into glass containers in batches using a 0.15mm sieve, with each batch having a thickness of 0.3mm. After each batch of sample was sieved, an inorganic arsenic additive solution was evenly sprayed onto the powder using an electric sprayer. The working pressure of the sprayer was set to 0.2MPa, the spray height was controlled at 20cm, the volume-to-mass ratio (m / v) of the solid powder to the spray solution was 1g:0.8mL, and the continuous spraying time for each spray coverage area was 5s.

[0076] S5. Place the powder added in S4 into a vertical mixer, stir at a speed of 50 r / min for 15 min, and then let it stand for 120 min to form a uniform moist powder sample.

[0077] S6. Place the damp powder sample after step S5 on a stainless steel tray, spread the sample into a thin layer (2cm), and place it in a forced-air drying oven to dry. The drying temperature program is as follows: heat up to 60℃, keep for 4h, then heat up to 105℃, keep for 0.5h.

[0078] S7. Place the dried sample from step S6 into a pulverizer, pulverize and sieve it. The sieve mesh size is 0.15 mm. Mix the undersize material with a high-efficiency mixer. The high-efficiency mixer has a mixing speed of 40 r / min and a mixing time of 8 h.

[0079] S8. The sample obtained in step S7 is sealed in a glass bottle with nitrogen filling. The nitrogen purity is ≥99.99%, the nitrogen filling pressure is 0.05MPa, the number of replacements is 3, and it is stored at 4℃ to obtain the standard quality control sample.

[0080] (iv) Verification of uniformity, long-term stability and accuracy

[0081] The testing of the standard quality control samples of this invention includes verification of homogeneity, long-term stability, and accuracy.

[0082] 1. Determination of inorganic arsenic in samples

[0083] (1) Test sample: Shiitake mushroom powder with added exogenous inorganic arsenic (pentavalent arsenic).

[0084] (2) Reagents: The experimental water meets the requirements of Grade I water in GB / T6682-2008 "Specifications and Test Methods for Water Used in Analytical Laboratories"; ammonium carbonate (superior grade), nitric acid (superior grade), methanol (chromatographic grade).

[0085] (3) Standard substances: Arsenite [As(III)] (GBW08666), Arsenate [As(V)] (GBW08667), Monomethylarsenic acid (MMA) (GBW08668), Dimethylarsenic acid (DMA) (GBW08669 0803), Arseniccholine (AsC) (GBW08667) and Arsenic betaine (AsB) (GBW08670) were all purchased from the National Institute of Metrology, China.

[0086] (4) Instruments and equipment

[0087] Ultrasonic cleaner (KQ2200E, Kunshan Ultrasonic Instrument Co., Ltd.); Inductively Coupled Plasma Tandem Mass Spectrometer (HPLC-ICP-MS / MS, Model: 8900, Agilent Technologies).

[0088] (5) Instrument conditions

[0089] Chromatographic conditions: Hamilton PRPX100 anion exchange column (4.1×250mm, 5μm); column temperature: 25℃; injection volume: 10μL; gradient elution program was used, mobile phase A was a 2% (v / v) methanol aqueous solution, mobile phase B was a 50mM ammonium carbonate ((NH4)2CO3) solution, and the elution program is shown in Table 2.

[0090] ICP-MS / MS conditions: RF power: 1500W; plasma gas (Ar) flow rate: 15L / min; reaction gas (O2) flow rate: 0.8mL / min; nebulizer gas flow rate: 1.0L / min. AsO2 was collected. 91+ Isotope data.

[0091] Table 2 Elution procedures for the separation of six arsenic speciations

[0092]

[0093] Sample pretreatment method: Weigh 0.2g of sample into a 15mL centrifuge tube and add 10mL of 0.1M HNO3 buffer. After mixing evenly with a vortex mixer, place the centrifuge tube into an ultrasonic extractor and extract at 60℃ for 1h. After extraction, centrifuge at 8000r / min for 3min, filter through a 0.45μm filter membrane, and then perform analysis.

[0094] Chromatograms of standard solutions and inorganic arsenic standard control samples (including other interfering arsenic forms) in edible fungi substrates are shown below. Figure 2 and Figure 3 ( Figure 2 This is the chromatogram of the standard solution. Figure 3The figure shows the chromatogram of the inorganic arsenic standard control sample prepared in the edible fungus matrix. As can be seen from the figure, the peaks of each target analyte in the prepared standard control sample are symmetrical and consistent with the corresponding standards. The target analyte peaks are well separated from other non-target impurity peaks, fully meeting the quality requirements of the control sample.

[0095] 2. Sample uniformity test

[0096] Test method: Ten samples were randomly selected from the pre-packaged samples. Each sample was tested twice independently under repeated conditions to determine the inorganic arsenic content (the sum of As(III) and As(V) content).

[0097] The test results and basic statistics are shown in Table 3. Uniformity was tested and evaluated using one-way ANOVA (F-test). The F-value was calculated using formulas (1) to (3), where Q1 is the between-group sum of squares, Q2 is the within-group sum of squares, the degrees of freedom are 9, the significance level α = 0.05, and the critical value of the degrees of freedom can be found in the table. 0.05 (9,10) = 3.02. Statistical calculation shows that the inorganic arsenic F = 1.93. The statistically calculated F value of inorganic arsenic is less than the critical value (3.02), indicating that the sample meets the homogeneity requirements and that the inorganic arsenic content in the sample is uniform.

[0098] Formula (1),

[0099] Formula (2),

[0100] Formula (3).

[0101] in, This is the group average. x ij For the first i The first in the group j One observation value; The total average of all samples; n The number of observations contained in each group; r Number of test groups ( r =2); K 1 For the degrees of freedom between groups, K 2 The degree of freedom within the group.

[0102] Table 3. Sample homogeneity test and calculation results (mg / kg)

[0103]

[0104] 3. Sample stability test

[0105] Stability tests were conducted on samples meeting homogeneity requirements. These tests included long-term stability (storage stability, conducted at 4°C) and short-term stability (transport stability, conducted at 30°C). Long-term stability was tested under conditions of resealing after opening (without nitrogen purging) for one year; short-term stability was tested under nitrogen purging conditions for seven days. Sampling followed a principle of starting with denser samples and gradually decreasing the density. Six sampling time points were set for both long-term and short-term stability tests. Three samples were randomly selected from each time point, and each sample underwent two parallel determinations. The average of the six determinations for the three samples was used to determine the inorganic arsenic content. In accordance with the "JJF 1343-2022 Standard Reference Material Determination and Homogeneity and Stability Assessment" standard, the t-test was used as the statistical method for stability testing. Curves showing the change of characteristic values ​​over time were plotted to assess whether there was a unidirectional trend in the sample's characteristic values. The stability of standard quality control samples is quantitatively evaluated based on a linear fitting model. Stability trend analysis is performed based on the model calculation results: when the |β1| value of a certain index is less than the critical value t·s(β1), the index is considered to remain stable during the observation period, indicating that the sample has good consistency. Specific calculations are shown in formulas (4) to (7):

[0106] Formula (4),

[0107] Formula (5),

[0108] Formula (6),

[0109] Formula (7);

[0110] in: β 1 The slope of the linear regression model; x i For time intervals; y i This is the actual test value; This is the average value over the time interval; This is the average of the actual measured values; S (β1) is the standard error of the slope; The value is predicted based on the regression equation; S r This represents the residual standard deviation.

[0111] Detailed test data and results are shown in Tables 4 and 5. After 12 months of observation, the |β1| value of inorganic arsenic in samples stored at 4℃ was 0.00229 (significantly less than the critical value t·s(β1) = 0.00475), confirming that this inorganic arsenic index exhibited good stability during the storage period (Table 4). For samples transported for a short period (7 days) at 30℃, the |β1| value of inorganic arsenic was 0.00174 (significantly less than the critical value t·s(β1) = 0.00446), confirming that this inorganic arsenic exhibited good stability during transportation (Table 5).

[0112] Table 4. Stability of inorganic arsenic in simulated samples under long-term storage at 4℃

[0113]

[0114] Table 5. Stability results of inorganic arsenic in simulated samples during transportation at 30℃.

[0115]

[0116] 4. Standard quality control sample value determination

[0117] In accordance with the technical specifications of JJF 1343-2022, "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials," standard values ​​were determined for the prepared samples. This invention employs a multi-laboratory joint assignment method, organizing eight accredited testing institutions (including provincial research institutes, universities, and certified third-party testing laboratories) to jointly conduct the assignment of the matrix standard quality control samples. In the data processing stage, the original test data were first subjected to a normality test; then, the Grubbs test was used to identify and remove outliers within each laboratory; simultaneously, the Cochrane test was used to evaluate the equiprecision characteristics of the measurement data between laboratories. Only when all statistical tests meet the specified requirements can the relevant data be considered statistically representative and included in the final statistical analysis of the assignment results.

[0118] The Shapiro-Wilke test was used to test the normality. From the table, we can find that W(n,p)=0.767 (where n=3, p=0.95). Table 6 shows the normality test results of inorganic arsenic from various laboratories. The result is 0.936, which is greater than 0.767. Therefore, it can be considered that the test data from various laboratories are normally distributed.

[0119] The Grubbs test was used to examine whether there were any suspicious values ​​in the test results within each laboratory group. According to the Grubbs critical value table, λ(0.05, 3) = 1.155. The results of inorganic arsenic in each laboratory in Table 6 show that the absolute value of the maximum residual value (Umax) of the test results is less than λ(0.05, 3) × S, indicating that there are no outliers and no suspicious values ​​among the test results of each laboratory. Therefore, all data are retained and can be used for statistical analysis of test results.

[0120] The Cochrane test was used to determine whether the results from different laboratories were of equal precision. The Cochrane test requires C to be less than or equal to C(α, m, n), indicating that the average values ​​of the data groups are of equal precision; otherwise, they are considered outliers and should be removed when calculating the final values. Referring to the criticality table, C(0.05, 8, 3) = 0.516. Table 6 shows that the C-value of the inorganic arsenic results from each laboratory is 0.2397, which is less than the critical value. Therefore, the data from each laboratory's test group are of equal precision, and all data should be retained for use in the statistical analysis of the final values.

[0121] Table 6. Interlaboratory detection data of inorganic arsenic in standard quality control samples.

[0122]

[0123] After removing suspicious values ​​and performing precision tests on the values ​​obtained from each laboratory, further significance and normality tests were conducted on the average values ​​of each laboratory. Statistical analysis showed no significant difference among the average values ​​of the eight laboratories (p>0.05), and the values ​​conformed to a normal distribution (p>0.05), indicating that the data met the requirements for statistical analysis. Therefore, in this example, the standard value of the inorganic arsenic standard control sample in the edible fungus substrate was determined to be the arithmetic mean of the average values ​​from each laboratory, and the standard value of inorganic arsenic (calculated as As) was set at 1.057 mg / kg.

[0124] According to JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", the uncertainty of the assignment result of the standard quality control sample consists of three parts: the uncertainty U caused by the non-homogeneity of the standard quality control sample. bb Uncertainty U caused by instability of standard quality control samples sts Uncertainty U caused by the standard quality control sample setting process char The combined uncertainty U can be determined by calculating the uncertainty of each part using formula (8). （y） At a confidence level of 95%, the expanded uncertainty U is twice the combined uncertainty (Formula 9):

[0125] Formula (8),

[0126] Formula (9).

[0127] Uncertainty U caused by non-uniformity of standard quality control samples bb Calculate according to formula (10):

[0128] Formula (10);

[0129] Among them, MS between Mean square between bottles (variance between groups);

[0130] MS within Mean square within the bottle (within-group variance);

[0131] n: The number of times each bottle is measured;

[0132] The raw data for the inter-bottle mean square and intra-bottle mean square calculations are provided in Table 3. The U0 of inorganic arsenic was calculated. bb The result was 0.0163 mg / kg.

[0133] U sts The calculation can be performed according to formula (11):

[0134] Formula (11);

[0135] Where R represents the interlaboratory range (maximum value - minimum value). The original data are provided in Table 6, and the U of inorganic arsenic was calculated. sts The result was 0.0242 mg / kg.

[0136] This invention uses joint determination by multiple laboratories (see Table 6), therefore U char The constant value formula is shown in formula (12):

[0137] Formula (12);

[0138] Wherein, the s-value is the standard deviation of the mean of the values ​​determined by the 8 laboratories, and the p-value is the total number of laboratories participating in the value determination, with a value of 8. The U-value of inorganic arsenic was calculated. char It is 0.00438 mg / kg.

[0139] Therefore, the total synthesis uncertainty of inorganic arsenic is calculated to be 0.0295 mg / kg, and the expanded uncertainty is 0.059 mg / kg. The characteristic value of the inorganic arsenic standard quality control sample in the edible fungus substrate in this invention example is 1.057 ± 0.059 mg / kg.

[0140] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a standard quality control sample of edible fungi substrate containing multiple arsenic forms, characterized in that, Includes the following steps: The dried edible fungi sample was made into powder to obtain a powder sample; the dried edible fungi sample was shiitake mushroom, porcini mushroom or matsutake mushroom, and the dried edible fungi sample contained monomethylarsine, dimethylarsine, arsenoside betaine and arsenite, and the content of arsenoside betaine in the dried edible fungi sample was not less than 0.01 mg / kg, the content of monomethylarsine was 0.01~0.1 mg / kg, the content of dimethylarsine was 0.01~0.1 mg / kg, and the content of arsenite was not less than 0.02 mg / kg; An aqueous solution of arsenate was added to the powder sample to obtain a moist powder sample; The wet powder sample was dried, pulverized and mixed in sequence to obtain the standard quality control sample of edible fungus substrate containing multiple arsenic forms. The content of inorganic arsenic in the standard quality control sample of edible fungus substrate containing multiple arsenic forms was 0.6~1.2 mg / kg. The contents of monomethylarsenic acid, dimethylarsenic acid, arsenic betaine, arsenite and inorganic arsenic were all calculated as elemental arsenic.

2. The preparation method according to claim 1, characterized in that, The method for preparing the powder includes: After removing the base of the stem from the dried edible fungi sample, it was cut into pieces, crushed, and sieved in sequence to obtain a powder sample; the sieve used for sieving had a mesh size of 0.15 mm.

3. The preparation method according to claim 1, characterized in that, The concentration of the arsenate aqueous solution is 0.5~1 mg / L, and the concentration is calculated as arsenic element; the mass ratio of the powder sample to the volume of the arsenate aqueous solution is 1 g:(0.8~1.2) mL.

4. The preparation method according to claim 1 or 3, characterized in that, The method of adding an aqueous solution of arsenate to the powder sample includes: The powder sample is laid up in a layer, and the arsenate aqueous solution is sprayed onto the layer. After stirring and standing, the moist powder sample is obtained.

5. The preparation method according to claim 4, characterized in that, The thickness of the layer is 0.3~0.6mm; the spraying pressure is 0.2~0.4MPa and the height is 20~40cm; the stirring rate is 30~100r / min and the time is 15~30min; the settling time is 120~240min.

6. The preparation method according to claim 1, characterized in that, The drying process includes sequentially performing a first drying and a second drying. The temperature of the first drying is 50-60°C and the time is 4-6 hours. The temperature of the second drying is 100-105°C and the time is 0.5-1 hours.

7. The preparation method according to claim 1, characterized in that, After crushing, the process further includes sieving the resulting crushed material through a sieve with a mesh size of 0.15 mm.

8. The preparation method according to claim 1, characterized in that, The mixing is carried out in a high-efficiency mixer with a working speed of 40~50 r / min and a mixing time of 6~8 h.

9. Standard quality control samples of edible fungi substrate containing multiple arsenic forms prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the standard quality control sample of edible fungi matrix containing multiple arsenic forms as described in claim 9 in the detection of inorganic arsenic content in edible fungi products.

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